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Nature Reviews Materials2017ReviewNon-viral Gene Delivery

Tools for translation: non-viral materials for therapeutic mRNA delivery

Khalid A. Hajj And Kathryn A. WhiteheadDOI 10.1038/natrevmats.2017.56

Summary

mRNA therapeutics have enormous potential for vaccines, cancer immunotherapy, protein replacement, and gene editing, but widespread clinical application is limited by the lack of safe and effective delivery vehicles. Naked mRNA is large (~10⁵–10⁶ Da), densely negatively charged, and rapidly degraded by nucleases; its cellular uptake rate is less than 1 in 10,000 molecules, and its intracellular half-life is only ~7 hours. There is a critical. Naked mRNA uptake: Cellular uptake rate of naked mRNA is <1 in 10,000 molecules; median intracellular half-life is only ~7 hours. - Endosomal escape bottleneck: Even world-class RNA delivery materials escape the.

Keywords

mRNALipid nanoparticleGene editingPolymericPolyethylenimineCancer immunotherapyT cells
Purpose: mRNA therapeutics have enormous potential for vaccines, cancer immunotherapy, protein replacement, and gene editing, but widespread clinical application is limited by the lack of safe and effective delivery vehicles. Naked mRNA is large (~10⁵–10⁶ Da), densely negatively charged, and rapidly degraded by nucleases; its cellular uptake rate is less than 1 in 10,000 molecules, and its intracellular half-life is only ~7 hours. There is a critical need for synthetic materials that can encapsulate, protect, and deliver mRNA payloads to the cytoplasm of target cells.
Hypothesis: As a review article, this work does not test a single hypothesis. Its central thesis is:

If synthetic non-viral materials (lipids, lipid-like materials, polymers, and hybrid systems) are rationally designed to encapsulate and protect IVT mRNA while overcoming extracellular and intracellular delivery barriers, then mRNA therapeutics can achieve efficacious protein expression in vivo and enable a broad range of clinical applications, including vaccines, cancer immunotherapy, protein replacement, and gene editing.

Aims: Primary Aim: To provide an overview of the field of mRNA therapeutics and describe recent advances in the development of synthetic materials that encapsulate and deliver mRNA payloads.
  • Secondary Aims:
  • To discuss the applications of mRNA drugs: protein replacement therapy, vaccines, cancer immunotherapy, and gene editing.
  • To outline the physical and chemical properties of mRNA and the barriers to systemic delivery.
  • To review non-viral delivery materials: lipids and lipid-like materials, polymers, and hybrid systems.
  • To discuss clinical translation of mRNA therapeutics and remaining challenges.
Delivery system:

Component: Payloads; Examples Discussed: In vitro-transcribed (IVT) mRNA encoding: antigens (influenza, Zika, HIV, rabies, Ebola), tumour-associated antigens (gp100, TRP2, ovalbumin), cytokines, antibodies (VRC01), gene-editing nucleases (Cas9), transcription factors (Yamanaka factors for iPSC), therapeutic proteins (factor IX, erythropoietin, VEGF-A, BMP2, BCL-2, FLT1), self-amplifying replicon mRNA

Component: Lipid and Lipid-Like Materials; Examples Discussed: Cationic lipids: DOTMA, DOTAP; Zwitterionic: DOPE; Ionizable lipids: DLin-MC3-DMA, C12-200, cKK-E12, TT3, OF-02, ZA3-EP10, L608; Helper lipids: DSPC, cholesterol; PEG-lipids; Lipid nanoparticles (LNPs); Lipid-like nanoparticles (LLNs)

Component: Polymers; Examples Discussed: Polyethylenimine (PEI, linear and branched), PEG-PAsp(DET), PEG-PAsp(TET), PEG-PAsp(TEP)-cholesterol, poly(glycoamidoamine) brushes (TarN3C10), PEGylated poly(β-amino esters), charge-altering releasable transporters (CARTs), cyclodextrin-PEI (CP 2k), PSA (polyethyleneimine-stearic acid)

Component: Hybrid Systems; Examples Discussed: Transcript-activated matrices (TAMs) with collagen sponge or fibrin gel/calcium phosphate granules, graphene oxide-PEI complexes, eIF4E-mRNA nanoplexes

Component: Routes of Administration; Examples Discussed: Intravenous, subcutaneous, intradermal, intramuscular, intranasal, intracerebral, intratracheal, intratumoral, hydrodynamic injection, local (knee joints, femoral bone)

Component: Target Cells/Tissues; Examples Discussed: Dendritic cells, T cells (CAR-T), hepatocytes, lung epithelium, neurons, tumour cells, bone, skeletal muscle, heart

Approach: This is a narrative review synthesizing preclinical and clinical literature. No primary experimental data are presented. The review covers:
  • In vitro studies: Cell lines including HeLa, NIH 3T3, U87, A549, HEK293, dendritic cells, primary fibroblasts.
  • In vivo animal models: Mice, rats, ferrets, pigs, cynomolgus monkeys, rhesus macaques.
  • Disease contexts: Haemophilia B, hereditary tyrosinaemia, hypercholesterolaemia, hepatitis B, osteoarthritis, Alzheimer's disease, fulminant hepatitis, pancreatic cancer, melanoma, Zika virus, influenza, HIV, Ebola, Toxoplasma gondii, bone defects, cardiovascular disease.
  • Clinical trials: Ex vivo transfection of dendritic cells and T cells; direct in vivo administration of mRNA-LNPs (Moderna, CureVac, Merck, AstraZeneca).
Key methods: As a review, the "methods" are literature synthesis and comparative analysis. Headline data cited from primary studies were generated using:
  • Reporter gene assays: Luciferase, GFP, erythropoietin (EPO).
  • Gene editing assays: CRISPR-Cas9 knockout (luciferase, Pcsk9), ZFN-mediated editing.
  • Immunogenicity assays: Antibody titres (IgG), neutralizing antibodies, cytotoxic T cell responses, cytokine induction (IFN-γ).
  • Biodistribution and pharmacokinetics: Luminescence imaging, tissue distribution, protein levels in serum.
  • Physicochemical characterization: Particle size, zeta potential, pKa, cryo-TEM.
  • Toxicity assessments: Liver damage, interferon response, hemolysis, cell viability.
  • Clinical trial endpoints: Safety, tolerability, immunogenicity, efficacy.
Key results: Naked mRNA uptake: Cellular uptake rate of naked mRNA is <1 in 10,000 molecules; median intracellular half-life is only ~7 hours. - Endosomal escape bottleneck: Even world-class RNA delivery materials escape the endosome only ~2% of the time. - Modified nucleotide enhancement: Incorporation of m¹ψ (N¹-methylpseudouridine) resulted in up to a 13-fold increase in gene expression compared with ψ or m5C/ψ in vivo. HPLC purification of IVT mRNA increased translation in primary cells up to 1,000-fold compared with unpurified mRNA. - Protein replacement (haemophilia B): LNPs containing 0.5 mg kg⁻¹ factor IX mRNA restored therapeutic factor IX levels within 12 hours after intravenous injection in knockout mice. Optimized LNPs with 1.1 mg kg⁻¹ human factor IX mRNA restored normal protein levels. - Zika vaccine: A single intradermal immunization of LNPs encapsulating 30 μg prM-E mRNA protected mice from Zika virus challenge at 2 weeks and 5 months post-injection. Non-human primates survived challenge 5 weeks after immunization with as little as 50 μg prM-E mRNA. - Influenza vaccine: Mice were protected from lethal virus following intradermal immunization with as little as 0.4 μg of mRNA; non-human primates developed robust immune responses. - Gene editing (CRISPR-Cas9): ZA3-EP10 LNPs facilitated 95% luciferase gene knockout in cell culture and potent gene editing in mouse liver, kidneys, and lungs following systemic delivery of 5 mg kg⁻¹ total RNA. TT3 LNPs mediated deletion of Pcsk9 in mouse livers. - Cancer immunotherapy: Subcutaneous injection of LNPs carrying gp100 and TRP2 mRNA resulted in transfection of antigen-presenting cells, reductions in tumour volume, and extended survival via CD8⁺ T cell activation. - CART polymers: Systemic delivery of luciferase mRNA mediated potent protein expression in liver and spleen, peaking 4 hours post-injection. - Translation kinetics: GFP production observed as early as 5 hours, with peak expression at 20 hours post-transfection with Lipofectamine 2000.
Interpretation: The authors conclude that mRNA holds significant promise as a genetic medicine, avoiding genomic integration risks associated with DNA-based gene therapy. The two overarching factors hindering clinical progression are IVT mRNA instability/immunogenicity and the lack of sufficiently efficacious delivery systems. The most clinically advanced materials are lipid nanoparticles. The authors emphasize that because most LNP formulations are proprietary, more academic studies are needed to understand how lipid structure and nanoparticle formulation affect efficacy. With the right delivery materials, mRNA therapeutics have the potential to revolutionize medicine, facilitating personalized, gene-based therapies.
Limitations: Limitations inherent to the review:
  • No primary experimental data; conclusions are synthesized from existing literature.
  • No systematic search strategy or meta-analysis.
  • Focus is primarily on non-viral synthetic materials; viral vectors are discussed but not exhaustively.
  • Most cited studies are preclinical or early clinical; limited late-stage clinical data.

Limitations of the field highlighted by the authors:

  • mRNA instability and immunogenicity: IVT mRNA is susceptible to degradation by exonucleases and endonucleases; unmodified mRNA triggers innate immune responses via RIG-I and TLRs.
  • Delivery barriers: Nanoparticles must evade immune cells and renal clearance, cross the endothelial barrier, diffuse through the extracellular matrix, mediate cellular uptake, and escape the endosome — the last being the most daunting, with only ~2% escape efficiency.
  • Cationic lipid toxicity: Cationic lipids (DOTAP, DOTMA) can cause liver damage, elicit interferon-γ responses, and be neutralized by anionic serum proteins.
  • Polymer toxicity: High molecular weight (>25 kDa) and highly branched PEI is toxic due to interaction with serum proteins, causing aggregation.
  • Proprietary formulations: Most clinically advanced LNP formulations are proprietary, limiting broader scientific understanding.
  • Cost and procurement: Commercial modified mRNA costs ~US$1 per μg; an mRNA dose of 1 mg kg⁻¹ to an average-sized mouse would cost approximately $20.
  • Translation kinetics: Most studies report peak protein translation within 6 hours, but kinetics vary with delivery vehicle, target cell type, and encoded protein; more extensive studies are needed.
  • Coding sequence effects: Alteration of a single nucleotide can affect protein folding and translation rate; rare codons reduce translation rate.
  • Ex vivo therapy limitations: Costly, invasive, and usually limited to targeting white blood cells; systemic delivery strategies are needed for broad clinical potential.
  • Limited clinical translation: Most mRNA clinical trials rely on ex vivo transfection and re-infusion of immune cells; in vivo delivery is still in early-phase trials.

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Tools for translation: non-viral materials for therapeutic mRNA delivery | Brilliant Blue Biosciences